TRANSPLANTATION

Depletion of autoreactive immunologic memory followed by autologoushematopoietic stem cell transplantation in patients with refractory SLE induceslong-term remission through de novo generation of a juvenile and tolerantimmune system*Tobias Alexander,1,2 *Andreas Thiel,1,3 Oliver Rosen,4 Gero Massenkeil,4 Arne Sattler,1 Siegfried Kohler,1 Henrik Mei,1,5

Hartmut Radtke,5 Erika Gromnica-Ihle,6 Gerd-Rudiger Burmester,2 †Renate Arnold,4 †Andreas Radbruch,1 and†Falk Hiepe1,2

1German Rheumatism Research Center, Berlin; 2Department of Rheumatology and Clinical Immunology, 3Regenerative Immunology and Aging,Berlin-Brandenburg Center for Regenerative Therapies, 4Department of Hematology and Oncology and 5Institute for Transfusion Medicine, ChariteUniversitatsmedizin Berlin, Berlin; and 6Rheuma-Klinik Berlin-Buch, Berlin, Germany

Clinical trials have indicated that immu-noablation followed by autologous hema-topoietic stem cell transplantation (ASCT)has the potential to induce clinical remis-sion in patients with refractory systemiclupus erythematosus (SLE), but themechanisms have remained unclear. Wenow report the results of a single-centerprospective study of long-term immunereconstitution after ASCT in 7 patientswith SLE. The clinical remissions ob-served in these patients are accompaniedby the depletion of autoreactive immuno-

logic memory, reflected by the disappear-ance of pathogenic anti–double-strandedDNA (dsDNA) antibodies and protectiveantibodies in serum and a fundamentalresetting of the adaptive immune system.The latter comprises recurrence ofCD31�CD45RA�CD4� T cells (recent thy-mic emigrants) with a doubling in abso-lute numbers compared with age-matchedhealthy controls at the 3-year follow-up(P � .016), the regeneration of thymic-derived FoxP3� regulatory T cells, andnormalization of peripheral T-cell recep-

tor (TCR) repertoire usage. Likewise, re-sponders exhibited normalization of thepreviously disturbed B-cell homeosta-sis with numeric recovery of the naiveB-cell compartment within 1 year afterASCT. These data are the first to demon-strate that both depletion of the autore-active immunologic memory and a pro-found resetting of the adaptive immunesystem are required to reestablishself-tolerance in SLE. This trial wasregistered at www.clinicaltrials.gov as#NCT00742300. (Blood. 2009;113:214-223)

Introduction

Systemic lupus erythematosus (SLE) is a systemic autoimmunedisease with heterogeneous clinical manifestations. It is character-ized by the generation of pathogenic antibodies directed against avariety of autoantigens, including nuclear and cytoplasmic anti-gens, such as double-stranded DNA (dsDNA), nucleosomes, andby complement activation.1 It is thought that, in geneticallysusceptible persons, an initial breakdown of peripheral tolerancepermits the activation of autoreactive lymphocytes, which thenpropagate autoimmune responses in a self-perpetuating pro-cess.2,3 We recently demonstrated that autoimmune reactions inlupus-prone mice (NZB/W) result in the generation of long-lived plasma cells, which secrete pathogenic autoantibodies andare resistant to conventional immunosuppression and B-celldepletion therapy.4,5

Whereas glucocorticoids and immunosuppressants amelioratemanifestations of SLE in many patients, current therapies areinsufficient to control the disease in a subset of patients, and theirclinical prognosis remains poor because of the development of vitalorgan failure, cumulative drug toxicity, and the increased risk ofcardiovascular disease and malignancy.6 Immunoablative chemo-therapy followed by autologous hematopoietic stem cell trans-

plantation (ASCT) has recently emerged as a promising experi-mental therapy for severely affected patients, providing themthe potential to achieve treatment-free, long-term remission.7,8

The rationale for applying ASCT to autoimmune diseases hasbeen the hope that immunoablation could eliminate inflammation-driving pathogenic cells from the immune system and thatregeneration of the patients’ immune system from hematopoieticprecursors could reestablish immunologic tolerance.9,10 So far,direct evidence for that is lacking, and no study has determinedwhether immunoablation and ASCT can actually “reset theimmunologic clock” in SLE.

Here we describe the long-term reconstitution of T- and B-cellsubsets and serologic changes in 7 patients with SLE for up to8 years after receiving immunoablation and ASCT, and show thatimmunoablation with high-dose chemotherapy, methylpred-nisolone, and antithymocyte globulin (ATG) efficiently depletesnaive and memory T and B cells and long-lived plasma cells,including those that are autoreactive. In addition, ASCT reactivatedthe thymus, leading to the development of a tolerant, “juvenile”adaptive immune system, which is reflected by long-term, treatment-free, clinical remissions.

Submitted July 11, 2008; accepted August 15, 2008. Prepublished online asBlood First Edition paper, September 29, 2008; DOI 10.1182/blood-2008-07-168286.

*T.A. and A.T. contributed equally to this work.

†R.A., A.R., and F.H. (all senior authors) contributed equally to this work.

An Inside Blood analysis of this article appears at the front of this issue.

The publication costs of this article were defrayed in part by page chargepayment. Therefore, and solely to indicate this fact, this article is herebymarked ‘‘advertisement’’ in accordance with 18 USC section 1734.

© 2009 by The American Society of Hematology

214 BLOOD, 1 JANUARY 2009 � VOLUME 113, NUMBER 1

Methods

Clinical trial protocol

Enrollment in the monocentric phase 1/2 clinical trial included a diagnosisof SLE according to American College of Rheumatology classificationcriteria11 and failure of remission despite treatment with 2 different standardimmunosuppressive therapies, including at least 6 cycles of intravenouscyclophosphamide at doses of 500 to 1000 mg/m2. A detailed description ofthe patients and trial design has been published previously.8 Peripheralblood stem cells were collected by leukapheresis after infusion of 2.0 g/m2

cyclophosphamide followed by daily granulocyte colony-stimulating factor(10 �g/kg; Amgen, Thousand Oaks, CA) beginning 72 hours aftercyclophosphamide infusion. The graft was enriched for CD34� cells byCliniMACS (Miltenyi Biotec, Bergisch Gladbach, Germany). Data on thepurity of CD34�-enriched hematopoietic stem cells are reported in Table 1.Immune ablation was achieved by 50 mg/kg per day of cyclophosphamideintravenously for 4 days (days �5 to �2) and 30 mg/kg per day of rabbitATG (Fresenius, Bad Homburg, Germany) intravenously for 3 days (days�4 to �2); 1 g of methylprednisolone was administered intravenously oneach day of ATG infusion. The study was approved by the responsibleethics committee and was conducted in conformity with European LeagueAgainst Rheumatism and European Group for Blood and Marrow Transplan-tation guidelines for blood and bone marrow stem cell transplantation.Seven patients with SLE (Table 2) gave informed consent and wereconsecutively enrolled in the study. This study was performed in accor-dance with the Declaration of Helsinki.

Clinical responses to ASCT

All 7 patients successfully completed the protocol treatment. Clinicalremission, defined as a Systemic Lupus Erythematosus Disease Activity

Index (SLEDAI) of less than 3 without immunosuppressive treatment oruse of antimalarials and with less than 7.5 mg of prednisolone daily, wasachieved in all 7 patients. One patient relapsed after being free of clinicalsymptoms for 18 months after transplantation, as described earlier12; hedied of SLE-related pulmonary embolism 38 months after ASCT. Anotherpatient died because of uncontrolled invasive central nervous systemaspergillosis 3 months after ASCT. The remaining 5 patients showed noclinical or serologic evidence of SLE activity during a median follow-up of60 months (range, 24-96 months).

Blood samples and cell preparation

Peripheral blood mononuclear cells (PBMCs) were isolated from heparin-ized blood by Ficoll-Hypaque density gradient centrifugation (GE Health-care, Little Chalfont, United Kingdom).

Flow cytometry

Absolute CD4� and CD19� lymphocyte numbers were calculated based onthe total lymphocyte count and the percentage of CD4� and CD19� cells, asidentified by flow cytometry using the BD Multitest panel (BD Biosciences,San Jose, CA). The following monoclonal antibodies (mAbs) were used forphenotypic analyses: anti-CD19-peridinin chlorophyll protein Cy5.5(SJ25C1), anti–CD20-phycoerythrin (PE; 2H7), and anti–IgD–fluoresceinisothiocyanate (FITC; IA6-2), anti–CD4–peridinin chlorophyll proteinCy5.5 (SK3), anti–CD31-PE (MEC13.3), anti–CD45RA-FITC (L48), andanti–CD45RO-allophycocyanin (APC; UCHL-1), all obtained from BDBiosciences. Anti–CD27-Cy5 (2E4) was conjugated to Cy5 (GE Health-care) according to the manufacturer’s instructions. Immunofluorescencestaining was performed by incubating PBMCs in the presence of mAbs in1% bovine serum albumin in phosphate-buffered saline on ice for 10 minutesafter blocking with 10 �g of human IgG for 10 minutes. Cells were washedbefore analysis on a FACSCalibur flow cytometer (BD Biosciences).

FoxP3 expression analysis was performed using freshly isolatedPBMCs stained with anti–CD4-FITC (TT1), anti–CD25-APC (2A3; BD),and anti–Foxp3-PE (PCH101) using the anti-human FoxP3 Staining Set(eBioscience, San Diego, CA) according to the manufacturer’s instructions.At least 2.5 � 104 CD4� T cells were acquired.

We performed analysis of T-cell receptor (TCR) V� expression onfreshly isolated peripheral blood CD4� T cells by 4-color flow cytometryusing 22 TCR V�-specific mononuclear antibodies (IOTest Beta Mark;Beckman Coulter, Fullerton, CA) as described recently.13 TCR designationsare according to Arden’s nomenclature.14 At least 2.5 � 104 CD4� T cellswere acquired. Normal ranges were established for each V� member basedon confidence intervals (CIs) of 97.5% determined in 20 healthy persons.

Table 1. Purity of the CD34�-enriched hematopoietic stem cellgrafts

Patientno.

Percentage ofCD34�

No. of infusedCD34�/kg

Percentage ofCD3�

No. of infusedCD3�/kg

1 92.2 3.0 � 106 0.48 1.6 � 104

2 89.3 2.4 � 106 0.52 1.4 � 104

3 99.1 6.1 � 106 0.05 1.0 � 104

4 98.0 4.2 � 106 0.05 1.0 � 104

5 97.5 2.6 � 106 0.02 0.5 � 104

6 95.3 2.0 � 106 0.03 0.6 � 104

7 78.6 2.4 � 106 0.03 0.4 � 104

Table 2. Demographic data and clinical features of patients

Patientno.

Sex/age, y Clinical manifestation

SLEDAIpretransplantation

Therapypretransplantation

Follow-up,mo Clinical outcome Current therapy

1 F/27 Nephritis, abdominal vasculitis,

polyserositis, APS, cytopenia

25 CY, AZA, HCQ, CSA, MMF 96 Clinical remission 2 mg prednisolone

2 F/48 Nephritis, peripheral

neuropathy, polyserositis,

ventricular arrhythmia

23 CY, AZA, HCQ, MTX, MMF 96 Clinical remission None

3 M/37 Nephritis WHO IV, ventricular

arrhythmia, polyserositis,

APS, cytopenia

30 CY, AZA, HCQ, CSA, MTX,

MMF

38 Relapse 18� mo, exitus

letalis 38� mo

—

4 F/24 Nephritis WHO V, seizures,

psychosis, polyserositis,

APS, cytopenia

28 CY, AZA, CSA 72 Clinical remission None

5 F/31 Nephritis WHO II, seizures,

psychosis, APS, cytopenia

26 CY, AZA, MTX 3 Exitus letalis 3� mo —

6 F/30 Nephritis WHO IIa, APS,

cytopenia

23 CY, AZA, MTX, HQC 48 Clinical remission 4 mg prednisolone

7 M/19 Nephritis, cerebritis, APS

cytopenia

19 CY, AZA, MTX, HCQ 24 Clinical remission 4 mg prednisolone

APS indicates antiphospholipid syndrome; CY, cyclophosphamide; AZA, azathioprine; HCQ, hydroxychloroquine; CSA, cyclosporine; MTX, methotrexate; MMF,mycophenolate mofetil; and —, not applicable.

AUTOREACTIVE MEMORY DEPLETION AFTER ASCT FOR SLE 215BLOOD, 1 JANUARY 2009 � VOLUME 113, NUMBER 1

Perturbations of V� families were considered to be significant in patientswhen they were outside of these normal intervals.

Bone marrow mononuclear cells (BM-MNCs) were collected by bonemarrow aspiration in 1 patient after ASCT and in 1 healthy volunteer fromthe femur after joint surgery. BM-MNCs were isolated by Ficoll-Hypaquedensity gradient centrifugation (GE Healthcare) and stained with anti–CD38-APC (HIT2; BD PharMingen, San Diego, CA) and anti–CD138-PE (B-B4;Chemicon International, Temecula, CA). Cells were washed before acquisi-tion (LSR II flow cytometer; BD Biosciences) and analysis (FlowJoSoftware; TreeStar, San Carlos, CA).

Stimulation assays

For in vitro lymphocyte stimulation assays, 1 mL freshly collectedheparinized peripheral blood was stimulated in the presence of 1 �g/mL�CD28 (clone 28.2; BD PharMingen) for 6 hours at 37°C with thefollowing antigens: 1 �g/mL Staphylococcus aureus enterotoxin B as thepositive control (Sigma Chemie, Deisenhofen, Germany), 20 �g/mLnucleosomes, as described previously,15 10 �g/mL SmD1 peptide, asdescribed previously,16 varicella zoster virus lysate and cytomegaly virus(CMV) lysate (both Biodesign International, Kennebunk, ME). Brefeldin A(Sigma Chemie) was added for the last 4 hours of stimulation. Erythrocyteswere lysed with FACS lysing solution and permeabilized with FACS-Perm2(both from BD PharMingen) according to the manufacturer’s instructions.Fixed cells were stained for 30 minutes at room temperature with thefollowing antibodies: anti–CD154-PE (TRAP1) or anti–CD69-PE (FN-50),anti–CD4-PerCp Cy5.5 (RPA-T4), anti–CD14-FITC (M5E2) and anti–interferon gamma (IFN-�)–APC (B27) (all purchased from BD PharMin-gen). At least 2 � 105 CD4� T cells were analyzed.

Serologic analysis

Antinuclear antibodies (ANAs) were assessed by indirect immunofluores-cence on HEp-2 cells. Anti-dsDNA antibodies were detected by Crithidialuciliae immunofluorescence and commercial enzyme-linked immunosor-bant assay (ELISA).

Statistical analysis

T- and B-lymphocyte subpopulation frequencies were calculated usingCellQuest software (BD Biosciences). A paired t test was used to compare

(per patient and immune parameter) pretransplantation and posttransplanta-tion data using Graph Pad Prism 4 software (version 4.03; Graph PadSoftware, San Diego, CA). Based on distributional assumptions, theMann-Whitney U test was used to compare data from patients treated byASCT with those from healthy controls and conventionally treated SLEpatients. All P values were 2-sided; statistical significance was set at� � 0.05.

Results

Engraftment and leukocyte recovery

The median time for recovery to 0.5 � 109/L neutrophils and 109/Lleukocytes in peripheral blood was 14 days. Platelets recovered tomore than 20 � 109/L by a median of 12 days. No patient receivedunselected backup stem cell support. All patients had significantlyreduced baseline lymphocyte counts (mean, 0.33 0.14 � 103

cells/�L), reflecting lupus activity or side effects of immunosuppres-sive therapy. As in patients with hematologic diseases treated with asimilar regimen,17 peripheral lymphocyte counts reconstitutedslowly after ASCT and were still slightly reduced at the 6-monthfollow-up (0.94 0.32 � 103 cells/�L). Mean absolute lympho-cyte counts were back to normal at the 1-year follow-up(1.11 0.29 � 103 cells/�L) and remained stable thereafter inresponding patients.

Increased naive T cells after posttransplantation immunereconstitution

In SLE, pathogenic T-cell functions are thought to be mediated byautoreactive memory or memory effector CD4� T cells. Elimina-tion of such cells in vivo by immunoablation is therefore presumedto ameliorate autoimmune inflammation. Conversely, thymic reac-tivation is presumably required to reestablish central tolerance andto generate natural FoxP3� regulatory T cells. To assess the effectof immunoablation and ASCT on the CD4� T-cell compartment,we first used the phenotypic markers CD45RA and CD45RO to

Figure 1. Recovery of CD4� T-cell subsets over timein SLE patients treated by immunoablation and ASCTversus levels in age-matched healthy controls.(A) CD45RO� CD45RA� CD4� T-cell frequencies (me-dian values) in patients (�, �) versus controls (hc, �,n � 28). The patient represented by closed symbols hada complete relapse 18 months after ASCT (patient 3);data after the flare (f) were excluded from statisticalconsiderations. A Mann-Whitney U test was used forgroup comparison; a paired t test was performed tocompare pre-ASCT data and corresponding post-ASCTdata (*P .05, **P .005, ***P .001). (B) Absolutecounts of CD45RA� CD45RO� naive (�) and CD45RO�

CD45RA� memory (f) CD4� T cells (median values andranges) in patients versus controls (hc, n � 28). (C) CD31expression on CD45RA� CD45RO� CD4� T cells (me-dian values) in patients versus controls (hc, n � 28).(D) Absolute counts of CD45RA� CD31� thymicnaiveCD4� T cells (median values) in patients versus controls(hc, n � 28).

216 ALEXANDER et al BLOOD, 1 JANUARY 2009 � VOLUME 113, NUMBER 1

discriminate between (CD45RA� CD45RO�) naive and (CD45RO�

CD45RA�) memory CD4� T cells.The longitudinal analysis of reconstituting naive and memory

CD4� T cells is shown in Figure 1. At baseline, patients displayed asignificant CD4� T-cell lymphopenia compared with age-matchedhealthy controls, which was attributable to both CD45RA� naive(median, 35/�L vs 288/�L, P .001) and CD45RO� memoryCD4� T cells (median, 51/�L vs 433/�L, P .001), reflecting thedisturbed T-cell homeostasis of active SLE (Figure 1B). In theregenerative phase, the memory phenotype (CD45RO�CD45RA�)was the predominant CD4� T-cell subset; there was a significantincrease in this subpopulation at 6 months after treatment com-pared with baseline (median, 73.4% vs 53.3%, P � .116; Figure1A) with a doubling of absolute counts (median, 121/�L vs 51/mL,P � .027; Figure 1B). Naive CD4� CD45RA� T-cell counts werelow or undeterminable at that time but later increased continuously,reaching complete recovery 24 months after ASCT with significanthigher values than before ASCT (median, 244/�L vs 35/�L,P � .014; Figure 1B). CD45RO� Th counts (Figure 1B) remainedsignificantly diminished until the 4-year follow-up.

Increased output of RTEs

To determine whether CD45RA�CD45RO�CD4� T cells in theregenerated immune system were homeostatically expanded periph-eral T cells or naive T cells newly generated in the thymus, weanalyzed their expression of CD31, a surrogate marker of recentthymic emigrants (RTEs).18 Six months after ASCT, 85.2% to98.8% (median, 89.7%) of the CD45RA�CD4� T cells in respond-ing patients coexpressed CD31; this was significantly more thanbefore ASCT in the same patients (median, 75.3%, P � .049) andin age-matched healthy controls (median, 64.2%, P .001; Figure1C). CD31 expression in CD45RA� CD4� T cells of the respond-ing patients remained at these high frequencies during the entireobservation period. Recovery of this T-cell subset to numberscomparable with healthy controls was completed between 12 and24 months after ASCT (Figure 1D), reaching on average 5.2 to 12.1times the baseline levels. Remarkably, the number of recent thymicemigrants continued to increase in responding patients; not before36 months after ASCT, they reached a plateau at twice the levelobserved in age-matched healthy controls (median, 435/�L vs164/�L, P � .016), and at 15 times the baseline levels (median,435/�L vs 29/�L, P � .031). The number of RTEs decreased onlyin the patient who suffered a relapse 18 months after transplantation.

The thymus contributes to the regeneration of FoxP3�

regulatory T cells

Regeneration of the regulatory CD4� T cell (Treg) compartmentwas evaluated after ASCT by identifying peripheral blood CD4�

T cells costaining brightly for CD25 and expressing intracellularFoxP3 (Figure 2A). Conventionally treated patients with activeSLE had significantly lower frequencies of peripheral FoxP3�

Tregs than normal controls (median, 5.5% vs 8.0%, P � .001), asillustrated in Figure 2B. Those with inactive SLE had comparable,if not higher, frequencies of FoxP3� CD4� T cells than the controls(median, 10.5% vs 8.0%, P � .048). FoxP3� CD4� T-cell frequen-cies in regenerated immunoablated ASCT patients at time pointsfrom 2 to 7 years after ASCT (as depicted in Figure 2A) were ashigh as those in normal controls (median, 9.4%, vs 8.0%, P � .229;Figure 2B). Overall, the absolute numbers of FoxP3� CD4� T cellswere similar in both groups (median, 62.8/�L vs 67.2/�L, P � .963;Figure 2C). However, the patients were heterogeneous with respect

to numbers of FoxP3� CD4� T cells. The later the follow-up date,the higher the patients’ peripheral FoxP3� CD4� T-cell count(161.8/�L in patient 1 at 7 years) and the lower the count in patientsanalyzed at earlier time points after ASCT (31.1/�L in patient 7 at2 years).

Thymic output generates a new and diverse TCR repertoire

CD4� T-cell diversity in the patients’ regenerating immune systemswas analyzed with a panel of TCR V�-specific monoclonalantibodies by flow cytometry, as recently described.19 At baseline,all patients analyzed (n � 4) showed significantly expanded TCRV�-expressing CD4� T cells (Table 3) in line with previousfindings on restricted TCR repertoires in active SLE.20 Early afterASCT (� 3 months), CD4� T cells in these patients still exhibited ahighly restricted TCR repertoire, however, with a different TCRV� family usage profile (Table 3). At the time point of assessment,patients showed no clinical signs of active infection or lupus flareexcept for 1 patient with an acute systemic herpes infection(HHV-6, patient 4). However, all patients contracted frequentinfections during the period of neutropenia shortly after ASCT.Along with the emergence of thymic naive CD31� T cells, theCD4� TCR repertoire gradually normalized within 1 year afterASCT (Table 3). Except for 2 patients showing transient TCR V�

Figure 2. Phenotypic analysis of FoxP3� Treg levels in 5 patients after ASCTcompared with those in healthy controls and conventionally treated SLEpatients. (A) CD25 and FoxP3 expression on CD4� T cells at indicated times afterASCT in 5 patients and 1 control. (B) Median FoxP3 expression levels in CD4� T cells(as determined by flow cytometry in panel A) in 5 patients versus 14 healthy controls,10 conventionally treated patients with active SLE (SLEDAI � 6), and 14 convention-ally treated with inactive SLE (SLEDAI 6). Group comparisons were performedusing the Mann-Whitney U test. (C) FoxP3� CD4� Tregs (median absolute counts, asdetermined by flow cytometry in panel A) in 5 patients versus 14 normal controls.A Mann-Whitney U test was used for group comparison.

AUTOREACTIVE MEMORY DEPLETION AFTER ASCT FOR SLE 217BLOOD, 1 JANUARY 2009 � VOLUME 113, NUMBER 1

expansion (patient 4, V�5.1 at 4 years and patient 6, V�12 at2 years) after ASCT, the TCR V� profiles of CD4� T cellsremained stable and heterogeneous throughout follow-up. At the3-year follow-up, the regenerated CD4� T-cell TCR V� familyusage was normal in all patients (Figure 3).

Early expansion of memory CD4� T cells is not driven byautoantigens

The specificity of CD4� memory T cells was analyzed by ex vivoshort-term restimulation of whole blood with viral antigens andautoantigens, and subsequent enumeration of reactivated T cells

expressing CD69 and IFN-� (Th1 memory cells) or CD154 (allmemory Th cells) during the early phase of immune reconstitu-tion.15,16,21 Notably, these ex vivo restimulation assays were performedin patients with identified viral infections based on clinical symptomsand corresponding serologic findings (patient 1: varicella-zoster virus,patient 4: human herpes virus 6, patient 6: CMV reactivation, patient 7:herpes simplex virus 1). During viral infections, Th effector memorycells specific for VZV (patient 1) and CMV (patient 6) were readilydetectable as CD69� or CD154� CD4� T cells coexpressing IFN-�.Inversely, T cells reacting to nucleosomes or SmD1 were not detectableearly after ASCT (Figure 4).

Table 3. Significantly expanded TCR V�-expressing CD4� T cells at baseline and during follow-up after ASCT in patients 4 to 7

Patient no.TCR

Beforetransplantation

3 months aftertransplantation

6 months aftertransplantation

1 year aftertransplantation

2 years aftertransplantation

3 years aftertransplantation

4 years aftertransplantation

4

BV5.1 5.5 8.1* 11.8* 8.9* 7.9 7.6 8.3*

BV5.2 1.7 2.4* 1.8 1.5 1.2 1.1 1.1

BV7.1 1.9 3.2* 1.9 2.7* 2.2 2.1 2.2

BV9 4.5 6.4* 4.3 3.5 3.0 3.3 2.9

BV13.6 1.5 6.7* 1.9 2.2 0.6 2.3 2.0

BV16 5.7* 0.5 0.3 0.6 0.2 1.1 0.7

5

BV14 8.4* — — — — — —

6

BV7.2 2.7* 1.7 1.4 1.9 1.6 1.4 2.0

BV12 2.1 2.3 2.5 2.2 2.7* 2.5 2.6

BV13.2 5.0* 3.5 2.9 3.4 3.4 3.1 3.5

BV20 3.7* 4.7* 1.1 1.5 2.1 1.4 2.1

7

BV4 2.6* 1.9 2.6* 2.2 2.3 — —

BV12 2.1 3.4* 3.2* 2.5 1.7 — —

BV13.1 5.2 12.9* 4.7 4.8 5.1 — —

— indicates not applicable.*Significant expansions according to criteria described in “Methods.”

Figure 3. CD4 TCR V� repertoires in 5 patients afterASCT. TCR V� family usage in peripheral blood CD4�

T cells (f) in 5 patients 3 years after treatment.� represent median values in 20 healthy donors; bound-aries indicate the 2.5th and 97.5th percentiles.

218 ALEXANDER et al BLOOD, 1 JANUARY 2009 � VOLUME 113, NUMBER 1

Normalization of disturbed B-cell homeostasis after ASCT

Active SLE is characterized by marked B lymphocytopenia, whichreportedly affects CD27� naive B cells more than CD27� memoryB cells.22 A prominent population of peripheral plasma blasts hasalso been observed in active disease.22,23 To evaluate the effect ofimmunoablation and ASCT on these B-cell disturbances, weanalyzed peripheral blood B lymphocytes from regenerating im-mune systems for IgD, CD27, and CD20 expression.22,24,25

Before treatment, patients had significantly lower numbers ofIgD� naive B cells than normal controls (median, 4/�L vs 202/�L,P .001; Figure 5) as well as a predominance of IgD� memoryB lymphocytes (median, 67.2% vs 23.5%, P � .003) and a promi-nent population of CD27high CD20� plasma blasts (median, 10.3%vs 0.9%, P � .006) in peripheral blood. After ASCT, B lympho-cytes predominantly displayed a naive IgD� phenotype. Completenumeric recovery of this subset was observed by 12 months afterASCT, with counts 50 times higher than at baseline (median,

Figure 4. In vitro whole-blood stimulation assays in4 patients early after ASCT. Levels of reactivated CD4�

T cells coexpressing CD69 or CD154 and intracellularIFN-� after ex vivo short-term restimulation of wholeblood for 6 hours with viral antigens, autoantigens, andwithout antigens as control (without [w/o]) in 4 patients atindicated time points after ASCT.

Figure 5. Recovery of CD19� B-cell subsets over timein SLE patients treated by immunoablation and ASCTversus levels in healthy controls. (A) Levels of IgD�

memory B cells among CD19� B cells (median values) inpatients (�, �) versus controls (hc, �, n � 32). Thepatient represented by closed symbols had a completerelapse 18 months after ASCT (patient 3); data after theflare (f) were excluded from statistical considerations.A Mann-Whitney U test was used for group comparison;a paired t test was performed to compare pre-ASCT dataand corresponding post-ASCT data (*P .05, **P .005,***P .001). (B) Absolute numbers of IgD� naive (�)and IgD� memory (f) CD19� B cells (median and range)in patients versus controls (hc, n � 32). A Mann-WhitneyU test was used for group comparison; a paired t test wasused to compare pre-ASCT data and correspondingpost-ASCT data. (C) Levels of CD27highCD20� plasmablasts among circulating CD19� B cells (median values)in patients versus controls (hc, n � 32). Dot plots showrepresentative examples in 1 patient (patient 6) at base-line and 6 months after ASCT. (D) Surface expression ofCD38 and CD138 on bone marrow mononuclear cells in1 patient (patient 7) early after ASCT (�1 month) and in1 healthy control.

AUTOREACTIVE MEMORY DEPLETION AFTER ASCT FOR SLE 219BLOOD, 1 JANUARY 2009 � VOLUME 113, NUMBER 1

196/�L vs 4/�L, P � .024). Absolute naive B-cell counts inresponders were well maintained throughout follow-up.

IgD� memory-phenotype B-cell frequencies drastically de-clined from a median of 67.2% at baseline to 7.0% within 6 monthsafter ASCT (P � .002). During immune regeneration, IgD� B-cellfrequencies remained lower than in healthy controls over the entirefollow-up period of up to 8 years (Figure 5A). IgD� memoryB-cell counts did not recover until 3 years after ASCT (Figure5B). IgD� memory B lymphocytes did not detectably expandbefore that time, except in the patient with the lupus flare(Figure 5A). CD20� CD27high plasma blast frequencies amongCD19� B cells normalized within 6 months after ASCT in allpatients analyzed, and normal levels persisted during the entirefollow-up period (Figure 5C).

Autoreactive and protective antibodies in serum are largelyextinguished after ASCT

All patients had ANAs and persistently high anti–double-stranded (anti-ds) DNA serum antibody titers before enrollment.After immunoablation and ASCT, anti-dsDNA antibodies disap-peared in all patients within 1 month (Table 4) and recurred onlyin the patient with reactivated disease (patient 3). Four of6 patients with a follow-up of at least 6 months after transplanta-tion showed a decrease in ANA titers to negative or 1:80, whichis regarded as clinically not significant (Table 4). From these4 patients, only 1 (patient 4) showed relevant ANA recurrence,which persisted from the 3-year follow-up onward withoutclinical symptoms of SLE. In 2 patients, ANA persisted, albeit insignificantly reduced titers.

Not only autoantibodies but also protective serum antibodiesspecific for measles, mumps, tetanus, and diphtheria disappeared inthe immunoablated patients. All patients had received the WorldHealth Organization-recommended vaccination before enrollment.Even though not all patients had reached protective levels ofvaccine-specific antibodies before immunoablation, significant

decreases in serum antibody levels for measles (P � .043, Figure6A), mumps (P � .028, Figure 6B), tetanus toxoid (P � .048,Figure 6C), and diphtheria (P � .049, Figure 6D) were observedwhen tested 1 or 2 years after ASCT (Figure 6). The serologic datapoint to an effective depletion of long-lived plasma cells from thebone marrow.5,26 A bone marrow aspiration sample from 1 patient(patient 7), obtained early after ASCT (�1 month), exhibitedalmost complete depletion of CD38� CD138� plasma cells with

Table 4. Titer of ANAs and anti–dsDNA antibodies before and after ASCT

Patient no./antibodies

Beforetransplantation

After ASCT

1month

6months

12months

24months

36months

48months

60months

72months

84months

96months

1

ANA 5120 320 Negative Negative 320 80 160 320 80 80 80

dsDNA 8 Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative

2

ANA 5120 320 80 80 80 80 80 160 80 80 80

dsDNA 64 Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative

3

ANA 2560 160 80 80 5120 2560 — — — — —

dsDNA 64 Negative Negative Negative Negative Negative — — — — —

4

ANA 20 480 640 Negative Negative Negative 320 1280 1280 1280 — —

dsDNA 64 Negative Negative Negative Negative Negative Negative Negative Negative — —

5

ANA 2560 320 — — — — — — — — —

dsDNA 32 Negative — — — — — — — — —

6

ANA 10 240 2560 2560 2560 2560 5120 5120 — — — —

dsDNA 80 Negative Negative Negative Negative Negative Negative — — — —

7

ANA 10 240 1280 1280 640 640 — — — — — —

dsDNA 80 Negative Negative Negative Negative — — — — — —

ANA indicates antinuclear antibodies, inverse titer; dsDNA, anti–double-stranded DNA antibodies (C luciliae assay), inverse titer; and —, not applicable.

Figure 6. Changes in protective antibody titers over time after ASCT. Protectiveserum antibody titers for (A) measles, (B) mumps, (C) tetanus, and (D) diphtheriadeclined significantly within 1 to 2 years after transplantation in all 6 patients with afollow-up of at least 6 months after transplantation. A paired t test was used tocompare pretransplantation and posttransplantation data.

220 ALEXANDER et al BLOOD, 1 JANUARY 2009 � VOLUME 113, NUMBER 1

only 0.03% among BM-MNC compared with a normal controlbone marrow with 1.24% of such cells (Figure 5D).

Discussion

Immunoablation followed by ASCT is an emerging treatmentoption for patients with severe autoimmune diseases refractory toconventional therapies, including SLE.7,10,27,28 In accordance withprevious reports,7,28 this regimen achieved long-term (up to 8 years)clinical and serologic treatment-free remissions in our lupuspatients. Notably, these patients originally had high disease activityand a poor prognosis, reflected by high SLEDAI scores andpersistent anti-dsDNA antibody titers. Although the clinical effi-cacy of this experimental therapy is rapidly becoming evident, itremains obscure how it works. Our detailed analysis of thelong-term reconstitution of the patients’ immune systems withrespect to the recurrence of T- and B-lymphocyte subsets and thecourse of serologic changes over time demonstrated successfuldepletion of autoreactive immunologic memory and the regenera-tion of a tolerant immune system from hematopoietic stem cells.Regeneration involved reactivation of the thymus and extensiverenewal of antigen-receptors, in other words, a “resetting of theimmunologic clock.”

Depletion of autoreactive immunologic memory by immunoa-blation was most drastically reflected in the complete disappear-ance of autoantibodies, particularly dsDNA-specific antibodies.Depletion of immunologic memory was not restricted to autore-active memory. In addition, pathogen-specific serum antibodiesfor mumps, measles, tetanus, and diphtheria were largelyextinguished. This drastic ablation of humoral memory suggeststhat the ATG used for immunoablation directly targets theplasma cells secreting these serum antibodies. It was recentlyshown that the plasma cells providing humoral memory arelong-lived cells that reside mostly in the bone marrow, wherethey dwell in specialized niches providing essential survivalsignals.5 Recent in vitro experiments indicated that polyclonalrabbit ATG directly targets plasma cells and B cells viacomplement-mediated lysis and apoptosis.29,30 In line with thishypothesis, we were able to stain plasma cells ex vivo with thepolyclonal rabbit ATG used for immunoablation (data notshown); moreover, plasma cells disappeared from bone marrow1 month after immunoablation in 1 case. The depletion oflong-lived plasma cells might be of particular relevance for thesuccess of immunoablative therapy. It has been demonstratedthat these plasma cells are resistant to immunosuppression bycyclophosphamide, irradiation, and CD20-mediated B-cell deple-tion.4,5,31 Hence, autoreactive long-lived plasma cells represent akey component of autoreactive immunologic memory. Persistentautoantibodies secreted by long-lived plasma cells could main-tain chronic inflammation and accelerate autoimmunity.32

A retrospective survey by the European Blood and MarrowTransplant and European League Against Rheumatism Registryrevealed that patients without complete loss of autoantibodyresponses after immunoablation and ASCT had higher rates ofrelapse.28 In our cohort, the only patient who relapsed becameanti-dsDNA-negative after immunoablation, but anti-Ro/SSAand anti-La/SSB antibodies persisted until the relapse. From theother patients, only 1 had anti-Ro/SSA antibodies beforeenrollment (patient 6). Similar to the patient with the relapse,anti-Ro/SSA antibodies persisted in this patient after ASCT,albeit without evidence for SLE reactivation. So far, it is not

clear why plasma cells secreting these autoantibodies seem to bemore resistant to the immunoablative regimen and if theirpersistence characterizes patients with a higher risk for relapse.

T-cell reconstitution after immunoablation was characterized bycontinued generation of new naive CD4� T cells for up to 8 yearsafter ASCT. In particular, naive CD45RA�CD4� T cells expressingCD31 with high overall clonal diversity of the CD4� TCRrepertoire were generated. These cells have been shown to berecent thymic emigrants.18 In the regenerated patients, absoluteCD45RA�CD31� naive CD4� T-cell counts continuously in-creased to levels twice as high as those in age-matched controls,resembling those in young children. This observation supports thenotion that, after immunoablation and ASCT, the naive CD4�

T-cell compartment is regenerated by thymic reactivation ratherthan by lymphopenic expansion of surviving naive T cells,emphasized earlier for patients undergoing immunoablation andASCT for treatment of hematologic malignancies33 and multiplesclerosis.34 In our SLE patients, the finding is even more relevant inlight of the disease- and treatment-related impairment of the naiveT-cell compartment, which has been attributed to intrinsic impair-ment of thymic export.35 Immunoablation and ASCT obviously cancorrect this deficiency, rejuvenate the CD4� T-cell compartment,and normalize naive T-cell homeostasis.

After monitoring the TCR V� family repertoire of the recurringCD4� T-cell compartment, we observed a drastic change in clonaldiversity of the TCR repertoire. The originally observed clonalexpansions and deletions disappeared, suggesting that treatmenthad led to the ablation of expanded clones and to the generation of acomplete repertoire of recent thymic emigrants.36

Among CD4� T cells, FoxP3� regulatory T cells regeneratedto frequencies and absolute numbers comparable with those innormal controls. The fact that regeneration of the Treg compart-ment was accompanied by the reappearance of naive T cells andrecent thymic emigrants suggests that these regulatory T cellswere generated in the thymus. Similar observations have beenmade in patients undergoing immunoablation and ASCT forjuvenile idiopathic arthritis, suggesting a common mechanismof action of stem cell transplantation in different autoimmunediseases.37

Whereas regeneration of thymic naive Th cells was delayedfor up to 1 year after ASCT, mature CD45RO� memory CD4�

T cells reappeared faster with on average a doubling of absolutecounts at 6 months after transplantation compared with baselinevalues. However, their TCR V� repertoires were highly re-stricted, reflecting responses to a limited array of availableantigens during lymphopenia.38,39 If peripheral T-cell expansionhad involved lymphopenia-driven proliferation of memoryT cells in response to low-affinity self-antigens, expansion ofautoreactive T-cell clones should have been observed.40 How-ever, we found no evidence of clonal expansion of autoreactiveT cells specific for SLE-associated autoantigens, such asnucleosomes or SmD1. This implies that the early expansion ofmemory CD4� T cells is not driven by autoantigens and, inparticular, not by those involved in SLE. Rather, we showed thatclonally expanded memory T cells reacted to virus-specificantigens in patients infected with specific viruses. This impli-cates protective pathogen-specific immune responses as a causeof clonal expansion of memory-phenotype T cells. The expan-sion of protective pathogen-specific T cells in response totreatment may contribute to the control of autoimmunity by

AUTOREACTIVE MEMORY DEPLETION AFTER ASCT FOR SLE 221BLOOD, 1 JANUARY 2009 � VOLUME 113, NUMBER 1

restricting the space available in the effector-memory compart-ment for autoreactive T cells, the expansion of which is drivenby (weak) reactions to autoantigens.

Regeneration of the B lymphocyte compartment in the treatedSLE patients resembled that of patients receiving ASCT fortreatment of hematologic malignancies.17,41 The majority of repopu-lating B cells initially showed a naive (IgD�) phenotype. Memory(IgD�) B cells did not reappear until later. In the present study, thisregeneration of the B-cell compartment was remarkable in view ofthe significant disturbances observed in our active SLE cohortbefore ASCT. These patients had shown naive B-cell lymphopenia,relative predominance of phenotypically memory B cells, andexpansion of CD27high CD20� plasma cell precursors. The com-plete normalization of these preexisting disturbances indicates thatimmunoablation had removed all autoreactive B cells. Apparently,the B-cell compartment also regenerates from stem cells afterimmunoablation and ASCT, and it is tolerant to self-antigens,including those that had been relevant in the patients beforetreatment.

In conclusion, this study provides direct evidence for a pro-found regeneration of the adaptive immune system in SLE patientsafter immunoablation and ASCT. All patients except 1 achievedlong-lasting clinical and serologic remissions and are no longerreliant on immunosuppressive therapy. The 1 exception relapsedafter having been in clinical remission for more than a year. Therelapse might be the result of insufficient ablation of autoreactiveimmunologic memory, presentation of tolerance-breaking autoanti-

gen forms to the regenerated immune system, or genetic predispo-sitions that restart the disease in the regenerated immune system.Our findings would propose that chronic autoimmunity is not anendpoint depending on continuous treatment with specific anti-inflammatory agents but may be cured by combining specifictargeting of autoreactive memory and effector cells with a reactiva-tion of thymic activity.

Acknowledgments

This work was supported by grants from the Bundesministeriumfur Bildung und Forschung (01GI9944/DRFZ C4.1) and theSonderforschungsbereiche (SFB) 650 TP12.

Authorship

Contribution: T.A., A.S., S.K., and H.M. did most of the experi-ments; F.H., R.A., A.T., and A.R. developed the concept anddesigned the clinical trial; T.A., O.R., G.M., H.R., E.G.-I., G.-R.B.,R.A., and F.H. conducted the clinical trial; and T.A., A.T., F.H., andA.R. wrote the manuscript.

Conflict-of-interest disclosure: The authors declare no compet-ing financial interests.

Correspondence: Tobias Alexander, Department of Rheumatologyand Clinical Immunology, Charite Universitatsmedizin, Chariteplatz 1,10117 Berlin, Germany; e-mail: tobias.alexander@charite.de.

References

1. Hahn BH. Antibodies to DNA. N Engl J Med.1998;338:1359-1368.

2. Lipsky PE. Systemic lupus erythematosus: anautoimmune disease of B cell hyperactivity. NatImmunol. 2001;2:764-766.

3. Shlomchik MJ, Craft JE, Mamula MJ. From T to Band back again: positive feedback in systemicautoimmune disease. Nat Rev Immunol. 2001;1:147-153.

4. Hoyer BF, Moser K, Hauser AE, et al. Short-livedplasmablasts and long-lived plasma cells contrib-ute to chronic humoral autoimmunity in NZB/Wmice. J Exp Med. 2004;199:1577-1584.

5. Radbruch A, Muehlinghaus G, Luger EO, et al.Competence and competition: the challenge ofbecoming a long-lived plasma cell. Nat Rev Im-munol. 2006;6:741-750.

6. Bernatsky S, Boivin JF, Joseph L, et al. Mortalityin systemic lupus erythematosus. ArthritisRheum. 2006;54:2550-2557.

7. Burt RK, Traynor A, Statkute L, et al. Nonmyeloa-blative hematopoietic stem cell transplantation forsystemic lupus erythematosus. JAMA. 2006;295:527-535.

8. Rosen O, Thiel A, Massenkeil G, et al. Autolo-gous stem-cell transplantation in refractory auto-immune diseases after in vivo immunoablationand ex vivo depletion of mononuclear cells. Ar-thritis Res. 2000;2:327-336.

9. Burt RK, Slavin S, Burns WH, Marmont AM. In-duction of tolerance in autoimmune diseases byhematopoietic stem cell transplantation: gettingcloser to a cure? Blood. 2002;99:768-784.

10. Sykes M, Nikolic B. Treatment of severe autoim-mune disease by stem-cell transplantation. Na-ture. 2005;435:620-627.

11. Hochberg MC. Updating the American College ofRheumatology revised criteria for the classifica-tion of systemic lupus erythematosus. ArthritisRheum. 1997;40:1725.

12. Rosen O, Hiepe F, Massenkeil G, Thiel A, Arnold

R. Relapse of systemic lupus erythematosus.Lancet. 2001;357:807-808.

13. MacIsaac C, Curtis N, Cade J, Visvanathan K.Rapid analysis of the Vbeta repertoire of CD4 andCD8 T lymphocytes in whole blood. J ImmunolMethods. 2003;283:9-15.

14. Arden B, Clark SP, Kabelitz D, Mak TW. HumanT-cell receptor variable gene segment families.Immunogenetics. 1995;42:455-500.

15. Bruns A, Blass S, Hausdorf G, Burmester GR,Hiepe F. Nucleosomes are major T and B cell au-toantigens in systemic lupus erythematosus. Ar-thritis Rheum. 2000;43:2307-2315.

16. Riemekasten G, Langnickel D, Ebling FM, et al.Identification and characterization of SmD183-119-reactive T cells that provide T cell help forpathogenic anti-double-stranded DNA antibodies.Arthritis Rheum. 2003;48:475-485.

17. Bomberger C, Singh-Jairam M, Rodey G, et al.Lymphoid reconstitution after autologous PBSCtransplantation with FACS-sorted CD34� he-matopoietic progenitors. Blood. 1998;91:2588-2600.

18. Kimmig S, Przybylski GK, Schmidt CA, et al.Two subsets of naive T helper cells with distinctT cell receptor excision circle content in humanadult peripheral blood. J Exp Med. 2002;195:789-794.

19. Muraro PA, Jacobsen M, Necker A, et al. Rapididentification of local T cell expansion in inflam-matory organ diseases by flow cytometric T cellreceptor Vbeta analysis. J Immunol Methods.2000;246:131-143.

20. Kolowos W, Gaipl US, Voll RE, et al. CD4 positiveperipheral T cells from patients with systemic lu-pus erythematosus (SLE) are clonally expanded.Lupus. 2001;10:321-331.

21. Frentsch M, Arbach O, Kirchhoff D, et al. Directaccess to CD4� T cells specific for defined anti-gens according to CD154 expression. Nat Med.2005;11:1118-1124.

22. Odendahl M, Jacobi A, Hansen A, et al. Disturbedperipheral B lymphocyte homeostasis in systemiclupus erythematosus. J Immunol. 2000;165:5970-5979.

23. Jacobi AM, Odendahl M, Reiter K, et al. Correla-tion between circulating CD27high plasma cellsand disease activity in patients with systemic lu-pus erythematosus. Arthritis Rheum. 2003;48:1332-1342.

24. Maurer D, Fischer GF, Fae I, et al. IgM and IgGbut not cytokine secretion is restricted to theCD27� B lymphocyte subset. J Immunol. 1992;148:3700-3705.

25. Klein U, Rajewsky K, Kuppers R. Human immu-noglobulin (Ig)M�IgD� peripheral blood B cellsexpressing the CD27 cell surface antigen carrysomatically mutated variable region genes: CD27as a general marker for somatically mutated(memory) B cells. J Exp Med. 1998;188:1679-1689.

26. Amanna IJ, Carlson NE, Slifka MK. Duration ofhumoral immunity to common viral and vaccineantigens. N Engl J Med. 2007;357:1903-1915.

27. Tyndall A, Saccardi R. Haematopoietic stemcell transplantation in the treatment of severeautoimmune disease: results from phase I/IIstudies, prospective randomized trials and fu-ture directions. Clin Exp Immunol. 2005;141:1-9.

28. Jayne D, Passweg J, Marmont A, et al. Autolo-gous stem cell transplantation for systemic lupuserythematosus. Lupus. 2004;13:168-176.

29. Zand MS, Vo T, Huggins J, et al. Polyclonal rabbitantithymocyte globulin triggers B-cell and plasmacell apoptosis by multiple pathways. Transplanta-tion. 2005;79:1507-1515.

30. Zand MS, Vo T, Pellegrin T, et al. Apoptosis andcomplement-mediated lysis of myeloma cells bypolyclonal rabbit antithymocyte globulin. Blood.2006;107:2895-2903.

222 ALEXANDER et al BLOOD, 1 JANUARY 2009 � VOLUME 113, NUMBER 1

31. Anolik JH, Barnard J, Cappione A, et al. Ritux-imab improves peripheral B cell abnormalities inhuman systemic lupus erythematosus. ArthritisRheum. 2004;50:3580-3590.

32. Manz RA, Moser K, Burmester GR, Radbruch A,Hiepe F. Immunological memory stabilizing auto-reactivity. Curr Top Microbiol Immunol. 2006;305:241-257.

33. Douek DC, Vescio RA, Betts MR, et al. Assess-ment of thymic output in adults after haematopoi-etic stem-cell transplantation and prediction ofT-cell reconstitution. Lancet. 2000;355:1875-1881.

34. Muraro PA, Douek DC, Packer A, et al. Thymicoutput generates a new and diverse TCR reper-toire after autologous stem cell transplantation in

multiple sclerosis patients. J Exp Med. 2005;201:805-816.

35. Kayser C, Alberto FL, da Silva NP, Andrade LE.Decreased number of T cells bearing TCR rear-rangement excision circles (TREC) in active re-cent onset systemic lupus erythematosus. Lupus.2004;13:906-911.

36. Berzins SP, Uldrich AP, Sutherland JS, et al. Thy-mic regeneration: teaching an old immune sys-tem new tricks. Trends Mol Med. 2002;8:469-476.

37. de Kleer I, Vastert B, Klein M, et al. Autologousstem cell transplantation for autoimmunity in-duces immunologic self-tolerance by reprogram-ming autoreactive T cells and restoring theCD4�CD25� immune regulatory network. Blood.2006;107:1696-1702.

38. Jameson SC. Maintaining the norm: T-cell ho-meostasis. Nat Rev Immunol. 2002;2:547-556.

39. Mackall CL, Bare CV, Granger LA, et al. Thymic-in-dependent T cell regeneration occurs via antigen-driven expansion of peripheral T cells resulting in arepertoire that is limited in diversity and prone toskewing. J Immunol. 1996;156:4609-4616.

40. King C, Ilic A, Koelsch K, Sarvetnick N. Homeo-static expansion of T cells during immune insuffi-ciency generates autoimmunity. Cell. 2004;117:265-277.

41. Avanzini MA, Locatelli F, Dos SC, et al. B lympho-cyte reconstitution after hematopoietic stem celltransplantation: functional immaturity and slowrecovery of memory CD27� B cells. Exp Hema-tol. 2005;33:480-486.

AUTOREACTIVE MEMORY DEPLETION AFTER ASCT FOR SLE 223BLOOD, 1 JANUARY 2009 � VOLUME 113, NUMBER 1